QS-21 is a complex saponin that has been used as a vaccine adjuvant, a property that has stimulated interest in this general class of compounds. In this collaboration, three complex saponins, Blighosides A, B, and C have been isolated from the tree Blighia sapida and their structures determined by high-resolution NMR. The molecular weights of the blighosides were obtained by mass spectrometry. Our results show that Blighoside A (MW 1086 Daltons) has an unbranched tetrasaccharide containing arabinose, rhamnose, glucose, and a second arabinose linked through one of the arabinose moieties to C-3 of a 30-carbon triterpene aglycone, hederagenin. The rhamnose is linked to C-2 of the first arabinose, the glucose to C-3 of the rhamnose, and the second arabinose to C-4 of the glucose, which is acetylated at O-3. Blighoside B (MW 1070 Daltons) contains the same tetrasaccharide moiety as Blighoside A, but linked to a slightly different triterpene, oleanolic acid, in which C-23 of hederagenin is now a methyl group instead of a hydroxymethyl group. Blighoside C (MW 1504 Daltons) has six monosaccharide units linked to oleanolic acid. Attached to C-3 of this triterpene is a linear hexasaccharide comprised of two xyloses, rhamnose, glucose, a second rhamnose, and another glucose. The second xylose is linked to C-3 of the first xylose, the first rhamnose to C-2 of the second xylose, the first glucose to C-3 of the preceding rhamnose, the second rhamnose to C-4 of the preceding glucose, and the final glucose to C-3 of the preceding rhamnose. Additionally, the first glucose is acetylated at O-3 and O-6, and the terminal glucose at O-4 and O-6. The NMR methods used for determination of structure included the use of 1D slices of 2D TOCSY data sets to generate separate sub-spectra for the individual monosaccharide residues, which allowed identification of sugar type by measurement of vicinal 1H1H coupling constants. Also used were 2D HMBC experiments that gave 1H/13C through-bond connectivities that defined nuclear neighbors in the structures, and also the sugar linkages and positions of acetyl substitution. 2D ROESY experiments indicated 1H/1H through-space interactions, especially for proximal protons in adjacent sugar residues. Additional resolution of the NMR signals of similar sugar residues was achieved by 2D 1H-coupled HSQC spectra, which also allowed identification of equatorial or axial protons in the sugar, as well as determination of the anomeric configuration of each sugar by measurement of their 1JC-1,H-1 coupling constants.